Antigen binding proteins that bind PD-L1

ABSTRACT

There is disclosed anti-PD-L1 IgG class antibodies that have an improved ability to be manufactured at higher yields. More specifically, there is disclosed human antibodies that bind PD-L1, PD-L1-binding fragments that can be manufactured at higher yields.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/288,912, filed on Jan. 29, 2016, the entire contents of which areexpressly incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 27, 2017, isnamed 126036-06602_ST25.txt and is 8.0 kilobytes in size.

TECHNICAL FIELD

The present disclosure provides anti-PD-L1 IgG class antibodies thathave an improved ability to be manufactured at higher yields. Morespecifically, the present disclosure provides human antibodies that bindPD-L1, PD-L1-binding fragments and derivatives of such antibodies, andPD-L1-binding polypeptides comprising such fragments.

BACKGROUND

Programmed death ligand 1 (PD-L1) is a 40 kDa type 1 transmembraneprotein. PD-L1 (human PD-L1 cDNA is composed of the base sequence shownby EMBL/GenBank Acc. No. NM_001267706 and mouse PD-L1 cDNA is composedof the base sequence shown by NM_021893) that is a ligand of PD-1 isexpressed in so-called antigen-presenting cells such as activatedmonocytes and dendritic cells. These cells present interaction moleculesthat induce a variety of immuno-inductive signals to T lymphocytes, andPD-L1 is one of these molecules that induce the inhibitory signal byligating PD-1. It has been revealed that PD-L1 ligation suppressed theactivation (cellular proliferation and induction of various cytokineproductions) of PD-1 expressing T lymphocytes. PD-L1 expression has beenconfirmed in not only immunocompetent cells but also a certain kind oftumor cell lines (cell lines derived from monocytic leukemia, cell linesderived from mast cells, cell lines derived from hepatic carcinomas,cell lines derived from neuroblasts, and cell lines derived from breastcarcinomas) (Nature Immunology (2001), vol. 2, issue 3, p. 261-267.).

Programmed death 1 (PD-1) is a member of the CD28 family of receptors,which includes CD28, CTLA-4, ICOS, PD-L1, and BTLA. The initial memberof the family, CD28, was discovered by functional effect on augmenting Tcell proliferation following the addition of monoclonal antibodies(Hutloff et al. (1999) Nature 397:263-266; Hansen et al. (1980)Immunogenics 10:247-260). Two cell surface glycoprotein ligands for PD-1have been identified, PD-L1 and PDL-2, and have been shown todown-regulate T cell activation and cytokine secretion occur uponbinding to PD-1 (Freeman et al. (2000) J. Exp. Med. 192:1027-34;Latchman et al. (2001) Nat. Immunol. 2:261-8; Carter et al. (2002) Eur.J. Immunol. 32:634-43; Ohigashi et al. (2005) Clin. Cancer Res.11:2947-53). Both PD-L1 (B7-H1) and PD-L2 (B7-DC) are B7 homologs thatbind to PD-1. Expression of PD-L1 on the cell surface has also beenshown to be upregulated through IFN-γ stimulation.

PD-L1 expression has been found in several murine and human cancers,including human lung, ovarian and colon carcinoma and various myelomas(Iwai et al. (2002) Proc. Natl. Acad. Sci. USA 99:12293-7; Ohigashi etal. (2005) Clin. Cancer Res. 11:2947-53). PD-L1 has been suggested toplay a role in tumor immunity by increasing apoptosis ofantigen-specific T-cell clones (Dong et al. (2002) Nat. Med. 8:793-800).It has also been suggested that PD-L1 might be involved in intestinalmucosal inflammation and inhibition of PD-L1 suppresses wasting diseaseassociated with colitis (Kanai et al. (2003) J. Immunol. 171:4156-63).

SUMMARY OF INVENTION

The present disclosure found that an antibody (called H6B1L) disclosedin U.S. Patent application US Patent Publication No. 2013-0323249 (Ser.No. 13/907,685 filed 31 May 2013) (the disclosure of which isincorporated by reference herein) as wild type SEQ ID NO. 213 for theheavy chain and SEQ ID NO. 214 for the light chain was not able to bemanufactured in sufficient quantity under certain conditions given aminoterminal fragmentation. Thus, the present disclosure provides variantH6B1L antibodies that can be manufactured, in particular, in CHO cellswithout light chain fragmentation.

In one embodiment, the present disclosure provides a fully humanantibody of an IgG class that binds to a PD-L1 epitope, which has aheavy chain variable domain sequence that is at least 95% identical tothe amino acid sequences of SEQ ID NO. 1, and that has a light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2 andSEQ ID NO. 3. Preferably, the fully human antibody has both a heavychain and a light chain wherein the antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2 (called H6B1L-EM herein) and SEQ ID NO. 1/SEQ IDNO. 3 (called H6B1L-EV herein).

In certain embodiments, the present disclosure provides a Fab fullyhuman antibody fragment, having a variable domain region from a heavychain and a variable domain region from a light chain, wherein the heavychain variable domain sequence that is at least 95% identical to theamino acid sequence SEQ ID NO. 1, and that has a light chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 2 and SEQ IDNO. 3. Preferably, the fully human antibody Fab fragment has both aheavy chain variable domain region and a light chain variable domainregion wherein the antibody has a heavy chain/light chain variabledomain sequence selected from the group consisting of SEQ ID NO. 1/SEQID NO. 2 and SEQ ID NO. 1/SEQ ID NO. 3.

In one embodiment, the present disclosure provides a single chain humanantibody, having a variable domain region from a heavy chain and avariable domain region from a light chain and a peptide linkerconnection the heavy chain and light chain variable domain regions,wherein the heavy chain variable domain sequence that is at least 95%identical to the amino acid sequence SEQ ID NO. 1, and that has a lightchain variable domain sequence that is at least 95% identical to theamino acid sequences selected from the group consisting of SEQ ID NO. 2and SEQ ID NO. 3. Preferably, the fully human single chain antibody hasboth a heavy chain variable domain region and a light chain variabledomain region, wherein the single chain fully human antibody has a heavychain/light chain variable domain sequence selected from the groupconsisting of SEQ ID NO. 1/SEQ ID NO. 2 and SEQ ID NO. 1/SEQ ID NO. 3.

In one embodiment, the present disclosure further provides a method fortreating a broad spectrum of mammalian cancers or a broad-spectrum ofinflammatory diseases and autoimmune diseases, comprising administeringan effective amount of an anti-PD-L1 polypeptide, wherein the anti-PD-L1polypeptide is selected from the group consisting of a fully humanantibody of an IgG class that binds to a PD-L1 epitope, a Fab fullyhuman antibody fragment, having a variable domain region from a heavychain and a variable domain region from a light chain, a single chainhuman antibody, having a variable domain region from a heavy chain and avariable domain region from a light chain and a peptide linkerconnection the heavy chain and light chain variable domain regions, andcombinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequence SEQID NO. 1, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 2 and SEQ ID NO. 3;

wherein the Fab fully human antibody fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequence SEQ ID NO. 1, and that has the light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2 and SEQ ID NO. 3; and

wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequence SEQ ID NO. 1, and that has the light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2 and SEQ ID NO. 3. Incertain embodiments, the heavy chain variable region comprises a CDR1domain, a CDR2 domain, and a CDR3 domain as set forth in the amino acidsequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7,

In certain embodiments, the fully human antibody has both a heavy chainand a light chain wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2 (called H6B1L-EM herein) and SEQ ID NO. 1/SEQ ID NO.3 (called H6B1L-EV herein). Preferably, the fully human antibody Fabfragment has both a heavy chain variable domain region and a light chainvariable domain region wherein the antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2 (called H6B1L-EM herein) and SEQ ID NO. 1/SEQ IDNO. 3 (called H6B1L-EV herein). Preferably, the fully human single chainantibody has both a heavy chain variable domain region and a light chainvariable domain region, wherein the single chain fully human antibodyhas a heavy chain/light chain variable domain sequence selected from thegroup consisting of SEQ ID NO. 1/SEQ ID NO. 2 and SEQ ID NO. 1/SEQ IDNO. 3.

In one embodiment, the invention provides a fully human antibody of anIgG class that binds to a PD-L1 epitope, wherein the antibody comprisesa heavy chain variable domain comprising an amino acid sequence that isat least 95% identical to the amino acid sequence set forth in SEQ IDNO. 1, and comprises a light chain variable domain comprising an aminoacid sequence as set forth in SEQ ID NO. 2 or SEQ ID NO. 3.

In other embodiments, the invention provides a fully human antibody ofan IgG class that binds to a PD-L1 epitope, wherein the antibodycomprises a heavy chain variable domain comprising a CDR1 domain, a CDR2domain, and a CDR3 domain as set forth in the amino acid sequences ofSEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, andcomprises a light chain variable domain comprising an amino acidsequence as set forth in SEQ ID NO. 2 or SEQ ID NO. 3.

The fully human antibody of the invention may, in certain embodiments,be an IgG1 or an IgG4.

In one embodiment, the invention also includes a pharmaceuticalcomposition comprising an anti-PD-L1 antibody (or fragment thereof) ofthe invention, and a pharmaceutically acceptable carrier.

The invention also includes a method for treating a human subject havingcancer or an autoimmune or inflammatory disease, said method comprisingadministering an effective amount of an anti-PD-L1 fully human antibody,Fab fragment, or scFv. In one embodiment, the broad spectrum ofmammalian cancers to be treated is selected from the group consisting ofovarian, colon, breast, lung cancers, myelomas, neuroblastic-derived CNStumors, monocytic leukemias, B-cell derived leukemias, T-cell derivedleukemias, B-cell derived lymphomas, T-cell derived lymphomas, mast cellderived tumors, and combinations thereof. In one embodiment, theautoimmune disease or inflammatory disease is selected from the groupconsisting of intestinal mucosal inflammation, wasting diseaseassociated with colitis, multiple sclerosis, systemic lupuserythematosus, viral infections, rheumatoid arthritis, osteoarthritis,psoriasis, Crohn's disease, and inflammatory bowel disease.

In one embodiment, the antibody or fragment of the invention is producedin a mammalian host cell, e.g., a Chinese hamster ovary (CHO) cell.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B shows a comparison mass spec peaks of the light chainsfor original wild type antibody H6B1L on the upper graph (FIG. 1A) andthe present variant antibody H6-B1L-EM on the lower graph (FIG. 1B). Asdescribed in FIG. 1B, no light chain (LC) fragment was detected for theH6B1LEM light chain in comparison to the parent light chain. Theantibodies described in FIG. 1 were produced in CHO cells.

DETAILED DESCRIPTION Definitions

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In an “intact immunoglobulin” (which has a heavy and lightchain structure like that of a naturally occurring immunoglobulin), eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa or lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. Seegenerally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). The variable regions of each light/heavy chain pair form theantibody binding site such that an intact immunoglobulin has two bindingsites. It should be noted that the term “naturally occurringimmunoglobulin” does not refer to the source of the immunoglobulin, butrather the overall structure of the immunoglobulin.

The variable regions of naturally occurring immunoglobulin chainsexhibit the same general structure of relatively conserved frameworkregions (FR) joined by three hypervariable regions, also calledcomplementarity determining regions or CDRs. From N-terminus toC-terminus, both light and heavy chains comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat et al. inSequences of Proteins of Immunological Interest, 5^(th) Ed., US Dept. ofHealth and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.Other numbering systems for the amino acids in immunoglobulin chainsinclude IMGT® (international ImMunoGeneTics information system; Lefrancet al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger andPluckthun, J. Mol. Biol. 309(3):657-670; 2001).

Antibodies can be obtained from sources such as serum or plasma thatcontain immunoglobulins having varied antigenic specificity. If suchantibodies are subjected to affinity purification, they can be enrichedfor a particular antigenic specificity. Such enriched preparations ofantibodies usually are made of less than about 10% antibody havingspecific binding activity for the particular antigen. Subjecting thesepreparations to several rounds of affinity purification can increase theproportion of antibody having specific binding activity for the antigen.Antibodies prepared in this manner are often referred to as“monospecific.” Monospecfic antibody preparations can be made up ofabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,99%, or 99.9% antibody having specific binding activity for theparticular antigen.

An “antibody” refers to an intact immunoglobulin or to an antigenbinding portion thereof that competes with the intact antibody forspecific binding, unless otherwise specified. In one embodiment, anantibody is an intact immunoglobulin. Antigen binding portions may beproduced by recombinant DNA techniques or by enzymatic or chemicalcleavage of intact antibodies. Antigen binding portions include, interalia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs), andcomplementarity determining region (CDR) fragments, single-chainantibodies (scFv), chimeric antibodies, diabodies, triabodies,tetrabodies, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified using the system described by Kabatet al. supra; Lefranc et al., supra and/or Honegger and Pluckthun,supra. One or more CDRs may be incorporated into a molecule eithercovalently or noncovalently to make it an antigen binding protein. Anantigen binding protein may incorporate the CDR(s) as part of a largerpolypeptide chain, may covalently link the CDR(s) to another polypeptidechain, or may incorporate the CDR(s) noncovalently. The CDRs permit theantigen binding protein to specifically bind to a particular antigen ofinterest.

An antigen binding protein may have one or more binding sites. If thereis more than one binding site, the binding sites may be identical to oneanother or may be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes an antibody, or antigen bindingfragment of an antibody, that has one or more variable and constantregions derived from human immunoglobulin sequences. In one embodiment,all of the variable and constant domains are derived from humanimmunoglobulin sequences (a fully human antibody). These antibodies maybe prepared in a variety of ways, examples of which are described below,including through the immunization with an antigen of interest of amouse that is genetically modified to express antibodies derived fromhuman heavy and/or light chain-encoding genes. Human antibodies may beidentified in a variety of ways, including through phage display orthrough immunization of a mouse with an antigen of interest where themouse is genetically modified to express antibodies derived from humanheavy and/or light chain-encoding genes. In one embodiment, a fullyhuman antibody is made using recombinant methods such that theglycosylation pattern of the antibody is different than an antibodyhaving the same sequence if it were to exist in nature.

A “humanized antibody” has a sequence that differs from the sequence ofan antibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In one embodiment, one or more of the CDRs are derivedfrom a human anti-PD-L1 antibody. In another embodiment, all of the CDRsare derived from a human anti-PD-L1 antibody. In another embodiment, theCDRs from more than one human anti-PD-L1 antibodies are mixed andmatched in a chimeric antibody. For instance, a chimeric antibody maycomprise a CDR1 from the light chain of a first human anti-PD-L1antibody, a CDR2 and a CDR3 from the light chain of a second humananti-PD-L1 antibody, and the CDRs from the heavy chain from a thirdanti-PD-L1 antibody. Other combinations are possible. Further, theframework regions may be derived from one of the same anti-PD-L1antibodies, from one or more different antibodies, such as a humanantibody, or from a humanized antibody. In one example of a chimericantibody, a portion of the heavy and/or light chain is identical with,homologous to, or derived from an antibody from a particular species orbelonging to a particular antibody class or subclass, while theremainder of the chain(s) is/are identical with, homologous to, orderived from an antibody (-ies) from another species or belonging toanother antibody class or subclass. Also included are fragments of suchantibodies that exhibit the desired biological activity (i.e., theability to specifically bind PD-L1).

A “Fab fragment” is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H1) domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H1) domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or VL domain (U.S. Pat. Nos. 6,846,634; 6,696,245, US App. Pub.20/0202512; 2004/0202995; 2004/0038291; 2004/0009507; 2003/0039958, andWard et al., Nature 341:544-546, 1989).

A “single-chain antibody” or an ‘scFv” is an antibody in which a V_(L)and a V_(H) region are joined via a linker (e.g., a synthetic sequenceof amino acid residues) to form a continuous protein chain. In certainembodiments, the linker is long enough to allow the protein chain tofold back on itself and form a monovalent antigen binding site (see,e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988,Proc. Natl. Acad. Sci. USA 85:5879-83). Diabodies are bivalentantibodies comprising two polypeptide chains, wherein each polypeptidechain comprises V_(H) and V_(L) domains joined by a linker that is tooshort to allow for pairing between two domains on the same chain, thusallowing each domain to pair with a complementary domain on anotherpolypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad.Sci. USA 90:6444-48, and Poljak et al., 1994, Structure 2:1121-23). Ifthe two polypeptide chains of a diabody are identical, then a diabodyresulting from their pairing will have two identical antigen bindingsites. Polypeptide chains having different sequences can be used to makea diabody with two different antigen binding sites. Similarly, tribodiesand tetrabodies are antibodies comprising three and four polypeptidechains, respectively, and forming three and four antigen binding sites,respectively, which can be the same or different.

A “neutralizing antibody” or an “inhibitory antibody” is an antibodythat inhibits the proteolytic activation of PD-L1 when an excess of theanti-PD-L1 antibody reduces the amount of activation by at least about20% using an assay such as those described herein in the Examples. Invarious embodiments, the antigen binding protein reduces the amount ofamount of proteolytic activation of PD-L1 by at least 30%, 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, and 99.9%.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specificationand using techniques known in the art. Preferred amino- andcarboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Computerized comparisonmethods can be used to identify sequence motifs or predicted proteinconformation domains that occur in other proteins of known structureand/or function. Methods to identify protein sequences that fold into aknown three-dimensional structure are known. See, Bowie et al., 1991,Science 253:164.

A “CDR grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of a particular species or isotype and theframework of another antibody of the same or different species orisotype.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human PD-L1) if it binds to the antigen with a dissociation constant of1 nanomolar or less.

An “antigen binding domain,” “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent identity” or “percent homology” of two polynucleotide ortwo polypeptide sequences is determined by comparing the sequences usingthe GAP computer program (a part of the GCG Wisconsin Package, version10.3 (Accelrys, San Diego, Calif.)) using its default parameters.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout and include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs (e.g., peptide nucleic acids andnon-naturally occurring nucleotide analogs), and hybrids thereof. Thenucleic acid molecule can be single-stranded or double-stranded. In oneembodiment, the nucleic acid molecules of the invention comprise acontiguous open reading frame encoding an antibody, or a fragment,derivative, mutein, or variant thereof.

Two single-stranded polynucleotides are “the complement” of each otherif their sequences can be aligned in an anti-parallel orientation suchthat every nucleotide in one polynucleotide is opposite itscomplementary nucleotide in the other polynucleotide, without theintroduction of gaps, and without unpaired nucleotides at the 5′ or the3′ end of either sequence. A polynucleotide is “complementary” toanother polynucleotide if the two polynucleotides can hybridize to oneanother under moderately stringent conditions. Thus, a polynucleotidecan be complementary to another polynucleotide without being itscomplement.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A nucleotide sequence is “operably linked” to a regulatory sequence ifthe regulatory sequence affects the expression (e.g., the level, timing,or location of expression) of the nucleotide sequence. A “regulatorysequence” is a nucleic acid that affects the expression (e.g., thelevel, timing, or location of expression) of a nucleic acid to which itis operably linked. The regulatory sequence can, for example, exert itseffects directly on the regulated nucleic acid, or through the action ofone or more other molecules (e.g., polypeptides that bind to theregulatory sequence and/or the nucleic acid). Examples of regulatorysequences include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Further examples of regulatorysequences are described in, for example, Goeddel, 1990, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

A “host cell” is a cell that can be used to express a nucleic acid,e.g., a nucleic acid of the invention. A host cell can be a prokaryote,for example, E. coli, or it can be a eukaryote, for example, asingle-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include the COS-7line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981,Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media (see Rasmussen et al.,1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient inDHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20),HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derivedfrom the African green monkey kidney cell line CV1 (ATCC CCL 70) (seeMcMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cellssuch as 293,293 EBNA or MSR 293, human epidermal A431 cells, humanColo205 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HL-60, U937, HaK or Jurkat cells. In one embodiment, ahost cell is a mammalian host cell which is not human. Typically, a hostcell is a cultured cell that can be transformed or transfected with apolypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein. In one embodiment, a host cell is a CHO cell.

The term “recombinant antibody” refers to an antibody that is expressedfrom a host cell (or cell line) transfected with an expression vector(or possibly more than one expression vector) comprising the codingsequence of the antibody, or a portion thereof (e.g., a DNA sequenceencoding a heavy chain or a light chain variable region as describedherein). In one embodiment, said coding sequence is not naturallyassociated with the cell. In one embodiment, a recombinant antibody hasa glycosylation pattern that is different than the glycosylation patternof an antibody having the same sequence if it were to exist in nature.In one embodiment, a recombinant antibody is expressed in a mammalianhost cell which is not a human host cell. Notably, individual mammalianhost cells have unique glycosylation patterns.

The terms “PD-L1 inhibitor” and “PD-L1 antagonist” are usedinterchangeably. Each is a molecule that detectably inhibits at leastone function of PD-L1. Conversely, a “PD-L1 agonist” is a molecule thatdetectably increases at least one function of PD-L1. The inhibitioncaused by a PD-L1 inhibitor need not be complete so long as it isdetectable using an assay. Any assay of a function of PD-L1 can be used,examples of which are provided herein. Examples of functions of PD-L1that can be inhibited by a PD-L1 inhibitor, or increased by a PD-L1agonist, include cancer cell growth or apoptosis (programmed celldeath), and so on. Examples of types of PD-L1 inhibitors and PD-L1agonists include, but are not limited to, PD-L1 binding polypeptidessuch as antigen binding proteins (e.g., PD-L1 inhibiting antigen bindingproteins), antibodies, antibody fragments, and antibody derivatives.

The terms “peptide,” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric.

A “variant” of a polypeptide (for example, an antibody) comprises anamino acid sequence wherein one or more amino acid residues are insertedinto, deleted from and/or substituted into the amino acid sequencerelative to another polypeptide sequence. Disclosed variants include,for example, fusion proteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified, e.g., via conjugation to anotherchemical moiety (such as, for example, polyethylene glycol or albumin,e.g., human serum albumin), phosphorylation, and glycosylation. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

PD-L1 Antigen Binding Proteins

The present disclosure provides a fully human antibody of an IgG classthat binds to a PD-L1 epitope with a binding affinity of 10⁻⁶M or less,that has a heavy chain variable domain sequence that is at least 95%identical to the amino acid sequence SEQ ID NO. 1, and that has a lightchain variable domain sequence that is at least 95% identical to theamino acid sequences selected from the group consisting of. Preferably,the fully human antibody has both a heavy chain and a light chainwherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2(called H6B1L-EM herein) and SEQ ID NO. 1/SEQ ID NO. 3 (called H6B1L-EVherein). Sequences of parent antibody H6B1L and its variants -EM and -EVare provided in the sequence table below. The -EM and -EV anti-PD-L1antibodies disclosed herein have been identified as having advantagesfor expression, particularly in CHO cells, wherein fragmentation isavoided.

Preferably, the anti-PD-L1 antibody (or fragment) of the invention bindsto human PD-L1.

Antigen binding proteins include fully human monoclonal antibodies thatinhibit a biological activity of PD-L1.

In some embodiments, the fully human antibody comprises a heavy chainvariable domain comprising a CDR3 domain (as determined using the Kabatnumbering scheme) having the amino acid sequence of SEQ ID NO: 7 and alight chain variable domain comprising a CDR3 domain having the aminoacid sequence of SEQ ID NO: 10. In some embodiments, the fully humanantibody comprises a heavy chain variable domain comprising a CDR2domain having the amino acid sequence of SEQ ID NO: 6 and a light chainvariable domain comprising a CDR2 domain having the amino acid sequenceof SEQ ID NO: 9. In some embodiments, the fully human antibody comprisesa heavy chain variable domain comprising a CDR1 domain having the aminoacid sequence of SEQ ID NO: 5 and a light chain variable domaincomprising a CDR1 domain having the amino acid sequence of SEQ ID NO: 8.In some embodiments, the fully human antibody comprises a heavy chainvariable domain comprising a CDR3, a CDR2 and a CDR1 having the aminoacid sequences of SEQ ID NOs: 7, 6, and 5, respectively, and a lightchain variable domain comprising a CDR3, a CDR2 and a CDR1 having theamino acid sequences of SEQ IDs NOs: 10, 9, and 8, respectively.

The invention provides, in certain embodiments, a fully human antibodyof an IgG class (e.g., IgG1 or IgG4) that binds to a PD-L1 epitope,wherein the antibody comprises a heavy chain variable domain comprisingan amino acid sequence that is at least 95% identical to the amino acidsequence set forth in SEQ ID NO. 1, and comprises a light chain variabledomain comprising an amino acid sequence as set forth in SEQ ID NO. 2 orSEQ ID NO. 3. In one embodiment, the invention provides a fully humanantibody of an IgG class (e.g., IgG1 or IgG4) that binds to a PD-L1epitope, wherein the antibody comprises a heavy chain variable domaincomprising a CDR1 domain, a CDR2 domain, and a CDR3 domain as set forthin the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ IDNO: 7, respectively, and comprises a light chain variable domaincomprising an amino acid sequence as set forth in SEQ ID NO. 2 or SEQ IDNO. 3.

Antibody fragments are also contemplated herein, wherein antibodyfragment binds to PD-L1 and comprises a heavy chain variable domaincomprising an amino acid sequence that is at least 95% identical to theamino acid sequence set forth in SEQ ID NO. 1, and comprises a lightchain variable domain comprising an amino acid sequence as set forth inSEQ ID NO. 2 or SEQ ID NO. 3. In one embodiment, the invention providesan anti-PD-L1 antibody fragment that comprises a heavy chain variabledomain comprising a CDR1 domain, a CDR2 domain, and a CDR3 domain as setforth in the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQID NO: 7, respectively, and comprises a light chain variable domaincomprising an amino acid sequence as set forth in SEQ ID NO. 2 or SEQ IDNO. 3.

The present disclosure provides a Fab fully human antibody fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical to the amino acid sequence SEQID NO. 1, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of. Preferably, the fully human antibody Fab fragment hasboth a heavy chain variable domain region and a light chain variabledomain region wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2 and SEQ ID NO. 1/SEQ ID NO. 3.

In some embodiments, the Fab fully human antibody fragment comprises aheavy chain variable domain comprising a CDR3 (as determined using theKabat numbering scheme) having the amino acid sequence of SEQ ID NO: 7and a light chain variable domain comprising a CDR3 having the aminoacid sequence of SEQ ID NO: 10. In some embodiments, the Fab fully humanantibody fragment comprises a heavy chain variable domain comprising aCDR2 having the amino acid sequence of SEQ ID NO: 6 and a light chainvariable domain comprising a CDR2 having the amino acid sequence of SEQID NO: 9. In some embodiments, the Fab fully human antibody fragmentcomprises a heavy chain variable domain comprising a CDR1 having theamino acid sequence of SEQ ID NO: 5 and a light chain variable domaincomprising a CDR1 having the amino acid sequence of SEQ ID NO: 8. Insome embodiments, the Fab fully human antibody fragment comprises aheavy chain variable domain comprising a CDR3, a CDR2 and a CDR1 havingthe amino acid sequences of SEQ ID NOs: 7, 6, and 5, respectively, and alight chain variable domain comprising a CDR3, a CDR2 and a CDR1 havingthe amino acid sequences of SEQ IDs NOs: 10, 9, and 8, respectively.

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques. Examples of such fragmentsinclude, but are not limited to, Fab and F(ab)₂ fragments.

The present disclosure provides a single chain human antibody, having avariable domain region from a heavy chain and a variable domain regionfrom a light chain and a peptide linker connection the heavy chain andlight chain variable domain regions, wherein the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequence SEQ ID NO. 1, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2 and SEQ ID NO. 3.Preferably, the fully human single chain antibody has both a heavy chainvariable domain region and a light chain variable domain region, whereinthe single chain fully human antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2 and SEQ ID NO. 1/SEQ ID NO. 3.

In some embodiments, the single chain human antibody comprises a heavychain variable domain comprising a CDR3 (as determined using the Kabatnumbering scheme) having the amino acid sequence of SEQ ID NO: 7 and alight chain variable domain comprising a CDR3 having the amino acidsequence of SEQ ID NO: 10. In some embodiments, the single chain humanantibody comprises a heavy chain variable domain comprising a CDR2having the amino acid sequence of SEQ ID NO: 6 and a light chainvariable domain comprising a CDR2 having the amino acid sequence of SEQID NO: 9. In some embodiments, the single chain human antibody comprisesa heavy chain variable domain comprising a CDR1 having the amino acidsequence of SEQ ID NO: 5 and a light chain variable domain comprising aCDR1 having the amino acid sequence of SEQ ID NO: 8. In someembodiments, the single chain human antibody comprises a heavy chainvariable domain comprising a CDR3, a CDR2 and a CDR1 having the aminoacid sequences of SEQ ID NOs: 7, 6, and 5, respectively, and a lightchain variable domain comprising a CDR3, a CDR2 and a CDR1 having theamino acid sequences of SEQ IDs NOs: 10, 9, and 8, respectively.

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers or inflammatory diseases or autoimmunediseases, comprising administering an effective amount of an anti-PD-L1polypeptide, wherein the anti-PD-L1 polypeptide is selected from thegroup consisting of a fully human antibody of an IgG class that binds toa PD-L1 epitope with a binding affinity of at least 10⁻⁶M, a Fab fullyhuman antibody fragment, having a variable domain region from a heavychain and a variable domain region from a light chain, a single chainhuman antibody, having a variable domain region from a heavy chain and avariable domain region from a light chain and a peptide linkerconnection the heavy chain and light chain variable domain regions, andcombinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequence SEQID NO. 1, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of; wherein the Fab fully human antibody fragment has theheavy chain variable domain sequence that is at least 95% identical tothe amino acid sequence SEQ ID NO. 1, and that has the light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2 andSEQ ID NO. 3; and wherein the single chain human antibody has the heavychain variable domain sequence that is at least 95% identical to theamino acid sequence SEQ ID NO. 1, and that has the light chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 2 and SEQ IDNO. 3.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2and SEQ ID NO. 1/SEQ ID NO. 3. Preferably, the fully human antibody Fabfragment has both a heavy chain variable domain region and a light chainvariable domain region wherein the antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2 and SEQ ID NO. 1/SEQ ID NO. 3. Preferably, thefully human single chain antibody has both a heavy chain variable domainregion and a light chain variable domain region, wherein the singlechain fully human antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2and SEQ ID NO. 1/SEQ ID NO. 3.

In particular embodiments, antigen binding proteins of the presentinvention have a binding affinity (K_(a)) for PD-L1 of at least 10⁶. Inother embodiments, the antigen binding proteins exhibit a K_(a) of atleast 10⁷, at least 10⁸, at least 10⁹, or at least 10¹⁰. In anotherembodiment, the antigen binding protein exhibits a K_(a) substantiallythe same as that of an antibody described herein in the Examples.

In another embodiment, the present disclosure provides an antigenbinding protein that has a low dissociation rate from PD-L1. In oneembodiment, the antigen binding protein has a K_(off) of 1×10⁻⁴ to ⁻¹ orlower. In another embodiment, the K_(off) is 5×10⁻⁵ to ⁻¹ or lower. Inanother embodiment, the K_(off) is substantially the same as an antibodydescribed herein. In another embodiment, the antigen binding proteinbinds to PD-L1 with substantially the same K_(off) as an antibodydescribed herein.

Affinity parameters may be determined using standard methods known inthe art, e.g., BIACORE.

In another aspect, the present disclosure provides an antigen bindingprotein that inhibits an activity of PD-L1. In one embodiment, theantigen binding protein has an IC₅₀ of 1000 nM or lower. In anotherembodiment, the IC₅₀ is 100 nM or lower; in another embodiment, the IC₅₀is 10 nM or lower. In another embodiment, the IC₅₀ is substantially thesame as that of an antibody described herein in the Examples. In anotherembodiment, the antigen binding protein inhibits an activity of PD-L1with substantially the same IC₅₀ as an antibody described herein.

In another aspect, the present disclosure provides an antigen bindingprotein that binds to human PD-L1 expressed on the surface of a celland, when so bound, inhibits PD-L1 signaling activity in the cellwithout causing a significant reduction in the amount of PD-L1 on thesurface of the cell. Any method for determining or estimating the amountof PD-L1 on the surface and/or in the interior of the cell can be used.In other embodiments, binding of the antigen binding protein to thePD-L1-expressing cell causes less than about 75%, 50%, 40%, 30%, 20%,15%, 10%, 5%, 1%, or 0.1% of the cell-surface PD-L1 to be internalized.

In another aspect, the present disclosure provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antigen binding protein has a half-life of fourdays or longer. In another embodiment, the antigen binding protein has ahalf-life of eight days or longer. In another embodiment, the antigenbinding protein is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antigen bindingprotein. In another embodiment, the antigen binding protein contains oneor more point mutations to increase serum half life, such as describedin WO00/09560, incorporated by reference herein.

The present disclosure further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that bind to two different epitopes of PD-L1, or to anepitope of PD-L1 and an epitope of another molecule, via two differentantigen binding sites or regions. Moreover, bispecific antigen bindingprotein as disclosed herein can comprise a PD-L1 binding site from oneof the herein-described antibodies and a second PD-L1 binding regionfrom another of the herein-described antibodies, including thosedescribed herein by reference to other publications. Alternatively, abispecific antigen binding protein may comprise an antigen binding sitefrom one of the herein described antibodies and a second antigen bindingsite from another PD-L1 antibody that is known in the art, or from anantibody that is prepared by known methods or the methods describedherein.

Numerous methods of preparing bispecific antibodies are known in theart. Such methods include the use of hybrid-hybridomas as described byMilstein et al., 1983, Nature 305:537, and chemical coupling of antibodyfragments (Brennan et al., 1985, Science 229:81; Glennie et al., 1987,J. Immunol. 139:2367; U.S. Pat. No. 6,010,902). Moreover, bispecificantibodies can be produced via recombinant means, for example by usingleucine zipper moieties (i.e., from the Fos and Jun proteins, whichpreferentially form heterodimers; Kostelny et al., 1992, J. Immunol.148:1547) or other lock and key interactive domain structures asdescribed in U.S. Pat. No. 5,582,996. Additional useful techniquesinclude those described in U.S. Pat. Nos. 5,959,083; and 5,807,706.

In another aspect, the antigen binding protein comprises a derivative ofan antibody. The derivatized antibody can comprise any molecule orsubstance that imparts a desired property to the antibody, such asincreased half-life in a particular use. The derivatized antibody cancomprise, for example, a detectable (or labeling) moiety (e.g., aradioactive, colorimetric, antigenic or enzymatic molecule, a detectablebead (such as a magnetic or electrodense (e.g., gold bead), or amolecule that binds to another molecule (e.g., biotin or streptavidin),a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, orpharmaceutically active moiety), or a molecule that increases thesuitability of the antibody for a particular use (e.g., administrationto a subject, such as a human subject, or other in vivo or in vitrouses). Examples of molecules that can be used to derivatize an antibodyinclude albumin (e.g., human serum albumin) and polyethylene glycol(PEG). Albumin-linked and PEGylated derivatives of antibodies can beprepared using techniques well known in the art. In one embodiment, theantibody is conjugated or otherwise linked to transthyretin (TTR) or aTTR variant. The TTR or TTR variant can be chemically modified with, forexample, a chemical selected from the group consisting of dextran,poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycolhomopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols and polyvinyl alcohols.

Oligomers that contain one or more antigen binding proteins may beemployed as PD-L1 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more antigen binding protein arecontemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple antigenbinding proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to the antigen binding proteins. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of antigen binding proteins attached thereto, asdescribed in more detail below.

In particular embodiments, the oligomers comprise from two to fourantigen binding proteins. The antigen binding proteins of the oligomermay be in any form, such as any of the forms described above, e.g.,variants or fragments. Preferably, the oligomers comprise antigenbinding proteins that have PD-L1 binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of Fusion Proteins Comprising CertainHeterologous Polypeptides Fused to Various Portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535; Byrn etal., 1990, Nature 344:677; and Hollenbaugh et al., 1992 “Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11.

One embodiment is directed to a dimer comprising two fusion proteinscreated by fusing a PD-L1 binding fragment of an anti-PD-L1 antibody tothe Fc region of an antibody. The dimer can be made by, for example,inserting a gene fusion encoding the fusion protein into an appropriateexpression vector, expressing the gene fusion in host cells transformedwith the recombinant expression vector, and allowing the expressedfusion protein to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yield thedimer.

The term “Fc polypeptide” includes native and mutein forms ofpolypeptides derived from the Fc region of an antibody. Truncated formsof such polypeptides containing the hinge region that promotesdimerization also are included. Fusion proteins comprising Fc moieties(and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

Another method for preparing oligomeric antigen binding proteinsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al., 1994, FEBS Letters 344:191. The use of amodified leucine zipper that allows for stable trimerization of aheterologous protein fused thereto is described in Fanslow et al., 1994,Semin. Immunol. 6:267-78. In one approach, recombinant fusion proteinscomprising an anti-PD-L1 antibody fragment or derivative fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomeric anti-PD-L1 antibody fragments or derivatives thatform are recovered from the culture supernatant.

Post-Translational Modifications of Polypeptides

In certain embodiments, the binding polypeptides of the invention mayfurther comprise post-translational modifications. Exemplarypost-translational protein modifications include phosphorylation,acetylation, methylation, ADP-ribosylation, ubiquitination,glycosylation, carbonylation, sumoylation, biotinylation or addition ofa polypeptide side chain or of a hydrophobic group. As a result, themodified soluble polypeptides may contain non-amino acid elements, suchas lipids, poly- or mono-saccharide, and phosphates. A preferred form ofglycosylation is sialylation, which conjugates one or more sialic acidmoieties to the polypeptide. Sialic acid moieties improve solubility andserum half-life while also reducing the possible immunogeneticity of theprotein. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76. Effectsof such non-amino acid elements on the functionality of a polypeptidemay be tested for its antagonizing role in PD-L1 or PD-1 function, e.g.,its inhibitory effect on angiogenesis or on tumor growth.

In one specific embodiment, modified forms of the subject solublepolypeptides comprise linking the subject soluble polypeptides tononproteinaceous polymers. In one specific embodiment, the polymer ispolyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes,in the manner as set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337.

PEG is a water soluble polymer that is commercially available or can beprepared by ring-opening polymerization of ethylene glycol according tomethods well known in the art (Sandler and Karo, Polymer Synthesis,Academic Press, New York, Vol. 3, pages 138-161). The term “PEG” is usedbroadly to encompass any polyethylene glycol molecule, without regard tosize or to modification at an end of the PEG, and can be represented bythe formula: X—O(CH₂CH₂O)_(n)-1CH₂CH₂OH (1), where n is 20 to 2300 and Xis H or a terminal modification, e.g., a C₁₋₄ alkyl. In one embodiment,the PEG of the invention terminates on one end with hydroxy or methoxy,i.e., X is H or CH₃ (“methoxy PEG”). A PEG can contain further chemicalgroups which are necessary for binding reactions; which results from thechemical synthesis of the molecule; or which is a spacer for optimaldistance of parts of the molecule. In addition, such a PEG can consistof one or more PEG side-chains which are linked together. PEGs with morethan one PEG chain are called multiarmed or branched PEGs. Branched PEGscan be prepared, for example, by the addition of polyethylene oxide tovarious polyols, including glycerol, pentaerythriol, and sorbitol. Forexample, a four-armed branched PEG can be prepared from pentaerythrioland ethylene oxide. Branched PEG are described in, for example, EP-A 0473 084 and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEGside-chains (PEG2) linked via the primary amino groups of a lysine(Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).

Although PEG is well-known, this is, to our knowledge, the firstdemonstration that a pegylated^(10F)n3 polypeptide can be pegylated andretain ligand binding activity. In a preferred embodiment, thepegylated^(10F)n3 polypeptide is produced by site-directed pegylation,particularly by conjugation of PEG to a cysteine moiety at the N- orC-terminus. Accordingly, the present disclosure provides atarget-binding ^(10F)n3 polypeptide with improved pharmacokineticproperties, the polypeptide comprising: a ^(10F)n3 domain having fromabout 80 to about 150 amino acids, wherein at least one of the loops ofsaid ^(10F)n3 domain participate in target binding; and a covalentlybound PEG moiety, wherein said ^(10F)n3 polypeptide binds to the targetwith a K_(D) of less than 100 nM and has a clearance rate of less than30 mL/hr/kg in a mammal. The PEG moiety may be attached to the ^(10F)n3polypeptide by site directed pegylation, such as by attachment to a Cysresidue, where the Cys residue may be positioned at the N-terminus ofthe ^(0F)n3 polypeptide or between the N-terminus and the mostN-terminal beta or beta-like strand or at the C-terminus of the ^(10F)n3polypeptide or between the C-terminus and the most C-terminal beta orbeta-like strand. A Cys residue may be situated at other positions aswell, particularly any of the loops that do not participate in targetbinding. A PEG moiety may also be attached by other chemistry, includingby conjugation to amines.

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski et al., J. Biol. Chem., 252, 3571 (1977) and J. Biol. Chem.,252, 3582 (1977), Zalipsky, et al., and Harris et. al., in:Poly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21and 22). It is noted that a binding polypeptide containing a PEGmolecule is also known as a conjugated protein, whereas the proteinlacking an attached PEG molecule can be referred to as unconjugated.

A variety of molecular mass forms of PEG can be selected, e.g., fromabout 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300), forconjugating to PD-L1-binding polypeptides. The number of repeating units“n” in the PEG is approximated for the molecular mass described inDaltons. It is preferred that the combined molecular mass of PEG on anactivated linker is suitable for pharmaceutical use. Thus, in oneembodiment, the molecular mass of the PEG molecules does not exceed100,000 Da. For example, if three PEG molecules are attached to alinker, where each PEG molecule has the same molecular mass of 12,000 Da(each n is about 270), then the total molecular mass of PEG on thelinker is about 36,000 Da (total n is about 820). The molecular massesof the PEG attached to the linker can also be different, e.g., of threemolecules on a linker two PEG molecules can be 5,000 Da each (each n isabout 110) and one PEG molecule can be 12,000 Da (n is about 270).

In a specific embodiment of the disclosure an PD-L1 binding polypeptideis covalently linked to one poly(ethylene glycol) group of the formula:—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR, with the —CO (i.e. carbonyl) of thepoly(ethylene glycol) group forming an amide bond with one of the aminogroups of the binding polypeptide; R being lower alkyl; x being 2 or 3;m being from about 450 to about 950; and n and m being chosen so thatthe molecular weight of the conjugate minus the binding polypeptide isfrom about 10 to 40 kDa. In one embodiment, a binding polypeptide's6-amino group of a lysine is the available (free) amino group.

The above conjugates may be more specifically presented by formula (II):P—NHCO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR (II), wherein P is the group of abinding polypeptide as described herein, (i.e. without the amino groupor amino groups which form an amide linkage with the carbonyl shown informula (II); and wherein R is lower alkyl; x is 2 or 3; m is from about450 to about 950 and is chosen so that the molecular weight of theconjugate minus the binding polypeptide is from about 10 to about 40kDa. As used herein, the given ranges of “m” have an orientationalmeaning. The ranges of “m” are determined in any case, and exactly, bythe molecular weight of the PEG group.

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on how the pegylated binding polypeptide will be usedtherapeutically, the desired dosage, circulation time, resistance toproteolysis, immunogenicity, and other considerations. For a discussionof PEG and its use to enhance the properties of proteins, see Katre,Advanced Drug Delivery Reviews 10: 91-114 (1993).

In one embodiment, PEG molecules may be activated to react with aminogroups on a binding polypeptide, such as with lysines (Bencham et al.,Anal. Biochem., 131, 25 (1983); Veronese et al., Appl. Biochem 11, 141(1985); Zalipsky et al., Polymeric Drugs and Drug Delivery Systems, adrs9-110 ACS Symposium Series 469 (1999); Zalipsky et al., Europ. Polym.J., 19, 1177-1183 (1983); Delgado et al., Biotechnology and AppliedBiochemistry, 12, 119-128 (1990)).

In one specific embodiment, carbonate esters of PEG are used to form thePEG-binding polypeptide conjugates. N,N′-disuccinimidylcarbonate (DSC)may be used in the reaction with PEG to form active mixedPEG-succinimidyl carbonate that may be subsequently reacted with anucleophilic group of a linker or an amino group of a bindingpolypeptide (see U.S. Pat. Nos. 5,281,698 and 5,932,462). In a similartype of reaction, 1,1′-(dibenzotriazolyl)carbonate anddi-(2-pyridyl)carbonate may be reacted with PEG to formPEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.5,382,657), respectively.

Pegylation of a ^(10F)n3 polypeptide can be performed according to themethods of the state of the art, for example by reaction of the bindingpolypeptide with electrophilically active PEGs (supplier: ShearwaterCorp., USA, www.shearwatercorp.com). Preferred PEG reagents of thepresent invention are, e.g., N-hydroxysuccinimidyl propionates(PEG-SPA), butanoates (PEG-SBA), PEG-succinimidyl propionate or branchedN-hydroxysuccinimides such as mPEG2-NHS (Monfardini et al., BioconjugateChem. 6 (1995) 62-69). Such methods may used to pegylated at an f-aminogroup of a binding polypeptide lysine or the N-terminal amino group ofthe binding polypeptide.

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson a binding polypeptide (Sartore et al., Appl. Biochem. Biotechnol.,27, 45 (1991); Morpurgo et al., Biocon. Chem., 7, 363-368 (1996);Goodson et al., Bio/Technology (1990) 8, 343; U.S. Pat. No. 5,766,897).U.S. Pat. Nos. 6,610,281 and 5,766,897 describes exemplary reactive PEGspecies that may be coupled to sulfhydryl groups.

In some embodiments where PEG molecules are conjugated to cysteineresidues on a binding polypeptide, the cysteine residues are native tothe binding polypeptide, whereas in other embodiments, one or morecysteine residues are engineered into the binding polypeptide. Mutationsmay be introduced into a binding polypeptide coding sequence to generatecysteine residues. This might be achieved, for example, by mutating oneor more amino acid residues to cysteine. Preferred amino acids formutating to a cysteine residue include serine, threonine, alanine andother hydrophilic residues. Preferably, the residue to be mutated tocysteine is a surface-exposed residue. Algorithms are well-known in theart for predicting surface accessibility of residues based on primarysequence or a protein. Alternatively, surface residues may be predictedby comparing the amino acid sequences of binding polypeptides, giventhat the crystal structure of the framework based on which bindingpolypeptides are designed and evolved has been solved (see Himanen etal., Nature. (2001) 20-27; 414(6866):933-8) and thus the surface-exposedresidues identified. In one embodiment, cysteine residues are introducedinto binding polypeptides at or near the N- and/or C-terminus, or withinloop regions.

In some embodiments, the pegylated binding polypeptide comprises a PEGmolecule covalently attached to the alpha amino group of the N-terminalamino acid. Site specific N-terminal reductive amination is described inPepinsky et al., (2001) JPET, 297, 1059, and U.S. Pat. No. 5,824,784.The use of a PEG-aldehyde for the reductive amination of a proteinutilizing other available nucleophilic amino groups is described in U.S.Pat. No. 4,002,531, in Wieder et al., (1979) J. Biol. Chem. 254,12579,and in Chamow et al., (1994) Bioconjugate Chem. 5, 133.

In another embodiment, pegylated binding polypeptide comprises one ormore PEG molecules covalently attached to a linker, which in turn isattached to the alpha amino group of the amino acid residue at theN-terminus of the binding polypeptide. Such an approach is disclosed inU.S. Patent Publication 2002/0044921 and in WO094/01451.

In one embodiment, a binding polypeptide is pegylated at the C-terminus.In a specific embodiment, a protein is pegylated at the C-terminus bythe introduction of C-terminal azido-methionine and the subsequentconjugation of a methyl-PEG-triarylphosphine compound via the Staudingerreaction. This C-terminal conjugation method is described in Cazalis etal., Bioconjug. Chem. 2004; 15(5):1005-1009.

Monopegylation of a binding polypeptide can also be produced accordingto the general methods described in WO 94/01451. WO 94/01451 describes amethod for preparing a recombinant polypeptide with a modified terminalamino acid alpha-carbon reactive group. The steps of the method involveforming the recombinant polypeptide and protecting it with one or morebiologically added protecting groups at the N-terminal alpha-amine andC-terminal alpha-carboxyl. The polypeptide can then be reacted withchemical protecting agents to selectively protect reactive side chaingroups and thereby prevent side chain groups from being modified. Thepolypeptide is then cleaved with a cleavage reagent specific for thebiological protecting group to form an unprotected terminal amino acidalpha-carbon reactive group. The unprotected terminal amino acidalpha-carbon reactive group is modified with a chemical modifying agent.The side chain protected terminally modified single copy polypeptide isthen deprotected at the side chain groups to form a terminally modifiedrecombinant single copy polypeptide. The number and sequence of steps inthe method can be varied to achieve selective modification at the N-and/or C-terminal amino acid of the polypeptide.

The ratio of a binding polypeptide to activated PEG in the conjugationreaction can be from about 1:0.5 to 1:50, between from about 1:1 to1:30, or from about 1:5 to 1:15. Various aqueous buffers can be used inthe present method to catalyze the covalent addition of PEG to thebinding polypeptide. In one embodiment, the pH of a buffer used is fromabout 7.0 to 9.0. In another embodiment, the pH is in a slightly basicrange, e.g., from about 7.5 to 8.5. Buffers having a pKa close toneutral pH range may be used, e.g., phosphate buffer.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated binding polypeptide, such as size exclusion(e.g. gel filtration) and ion exchange chromatography. Products may alsobe separated using SDS-PAGE. Products that may be separated includemono-, di-, tri- poly- and un-pegylated binding polypeptide, as well asfree PEG. The percentage of mono-PEG conjugates can be controlled bypooling broader fractions around the elution peak to increase thepercentage of mono-PEG in the composition. About ninety percent mono-PEGconjugates represents a good balance of yield and activity. Compositionsin which, for example, at least ninety-two percent or at leastninety-six percent of the conjugates are mono-PEG species may bedesired. In an embodiment of this invention the percentage of mono-PEGconjugates is from ninety percent to ninety-six percent.

In one embodiment, PEGylated binding polypeptide of the inventioncontain one, two or more PEG moieties. In one embodiment, the PEGmoiety(ies) are bound to an amino acid residue which is on the surfaceof the protein and/or away from the surface that contacts the targetligand. In one embodiment, the combined or total molecular mass of PEGin PEG-binding polypeptide is from about 3,000 Da to 60,000 Da,optionally from about 10,000 Da to 36,000 Da. In a one embodiment, thePEG in pegylated binding polypeptide is a substantially linear,straight-chain PEG.

In one embodiment, the PEG in pegylated binding polypeptide is nothydrolyzed from the pegylated amino acid residue using a hydroxylamineassay, e.g., 450 mM hydroxylamine (pH 6.5) over 8 to 16 hours at roomtemperature, and is thus stable. In one embodiment, greater than 80% ofthe composition is stable mono-PEG-binding polypeptide, more preferablyat least 90%, and most preferably at least 95%.

In another embodiment, the pegylated binding polypeptides of theinvention will preferably retain at least 25%, 50%, 60%, 70%, 80%, 85%,90%, 95% or 100% of the biological activity associated with theunmodified protein. In one embodiment, biological activity refers to itsability to bind to PD-L1, as assessed by KD, k_(on) or k_(off). In onespecific embodiment, the pegylated binding polypeptide protein shows anincrease in binding to PD-L1 relative to unpegylated bindingpolypeptide.

The serum clearance rate of PEG-modified polypeptide may be decreased byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative tothe clearance rate of the unmodified binding polypeptide. ThePEG-modified polypeptide may have a half-life (t_(1/2)) which isenhanced relative to the half-life of the unmodified protein. Thehalf-life of PEG-binding polypeptide may be enhanced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,250%, 300%, 400% or 500%, or even by 1000% relative to the half-life ofthe unmodified binding polypeptide. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf-life, such as the half-life of the protein in the serum or otherbodily fluid of an animal.

Therapeutic Methods, Formulations and Modes of Administration

Certain methods provided herein comprise administering a PD-L1 bindingantigen binding protein to a subject, thereby reducing a PD-L1-inducedbiological response that plays a role in a particular condition. Inparticular embodiments, methods of the invention involve contactingendogenous PD-L1 with a PD-L1 binding antigen binding protein, e.g., viaadministration to a subject or in an ex vivo procedure.

The term “treatment” encompasses alleviation or prevention of at leastone symptom or other aspect of a disorder, or reduction of diseaseseverity, and the like. An antigen binding protein need not effect acomplete cure, or eradicate every symptom or manifestation of a disease,to constitute a viable therapeutic agent. As is recognized in thepertinent field, drugs employed as therapeutic agents may reduce theseverity of a given disease state, but need not abolish everymanifestation of the disease to be regarded as useful therapeuticagents. Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a condition in order toconstitute a viable prophylactic agent. Simply reducing the impact of adisease (for example, by reducing the number or severity of itssymptoms, or by increasing the effectiveness of another treatment, or byproducing another beneficial effect), or reducing the likelihood thatthe disease will occur or worsen in a subject, is sufficient. Oneembodiment of the invention is directed to a method comprisingadministering to a patient a PD-L1 antagonist in an amount and for atime sufficient to induce a sustained improvement over baseline of anindicator that reflects the severity of the particular disorder.

The present disclosure features methods for treating conditions orpreventing pre-conditions which respond to an inhibition of PD-L1biological activity. Preferred examples are conditions that arecharacterized by inflammation or cellular hyperproliferation. Techniquesand dosages for administration vary depending on the type of specificpolypeptide and the specific condition being treated but can be readilydetermined by the skilled artisan. In general, regulatory agenciesrequire that a protein reagent to be used as a therapeutic is formulatedso as to have acceptably low levels of pyrogens. Accordingly,therapeutic formulations will generally be distinguished from otherformulations in that they are substantially pyrogen free, or at leastcontain no more than acceptable levels of pyrogen as determined by theappropriate regulatory agency (e.g., FDA).

As is understood in the pertinent field, pharmaceutical compositionscomprising the antibodies and fragments thereof of the disclosure areadministered to a subject in a manner appropriate to the indication.Thus, the invention includes a pharmaceutical composition comprising ananti-PD-L1 antibody (or fragment) disclosed herein, and apharmaceutically acceptable carrier.

Pharmaceutical compositions may be administered by any suitabletechnique, including but not limited to, parenterally, topically, or byinhalation. If injected, the pharmaceutical composition can beadministered, for example, via intra-articular, intravenous,intramuscular, intralesional, intraperitoneal or subcutaneous routes, bybolus injection, or continuous infusion. Localized administration, e.g.at a site of disease or injury is contemplated, as are transdermaldelivery and sustained release from implants. Delivery by inhalationincludes, for example, nasal or oral inhalation, use of a nebulizer,inhalation of the antagonist in aerosol form, and the like. Otheralternatives include eyedrops; oral preparations including pills,syrups, lozenges or chewing gum; and topical preparations such aslotions, gels, sprays, and ointments.

Advantageously, antigen binding proteins are administered in the form ofa composition comprising one or more additional components such as aphysiologically acceptable carrier, excipient or diluent. Optionally,the composition additionally comprises one or more physiologicallyactive agents, for example, a second inflammation- or immune-inhibitingsubstance, an anti-angiogenic substance, an analgesic substance, etc.,non-exclusive examples of which are provided herein. In variousparticular embodiments, the composition comprises one, two, three, four,five, or six physiologically active agents in addition to a PD-L1binding antigen binding protein.

Therapeutic compositions of the present disclosure may be administeredwith a pharmaceutically acceptable diluent, carrier, or excipient, inunit dosage form. Administration may be parenteral (e.g., intravenous,subcutaneous), oral, or topical, as non-limiting examples. In addition,any gene therapy technique, using nucleic acids encoding thepolypeptides of the invention, may be employed, such as naked DNAdelivery, recombinant genes and vectors, cell-based delivery, includingex vivo manipulation of patients' cells, and the like.

The composition can be in the form of a pill, tablet, capsule, liquid,or sustained release tablet for oral administration; or a liquid forintravenous, subcutaneous or parenteral administration; gel, lotion,ointment, cream, or a polymer or other sustained release vehicle forlocal administration.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Formulations for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

The polypeptide may be optionally administered as a pharmaceuticallyacceptable salt, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andanti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc). Formulations for oraluse may also be provided as chewable tablets, or as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. In general, the polypeptideis administered at about 0.01 μg/kg to about 50 mg/kg per day,preferably 0.01 mg/kg to about 30 mg/kg per day, most preferably 0.1mg/kg to about 20 mg/kg per day. The polypeptide may be given daily(e.g., once, twice, three times, or four times daily) or preferably lessfrequently (e.g., weekly, every two weeks, every three weeks, monthly,or quarterly). In addition, as is known in the art, adjustments for ageas well as the body weight, general health, sex, diet, time ofadministration, drug interaction, and the severity of the disease may benecessary, and will be ascertainable with routine experimentation bythose skilled in the art.

The PD-L1 binding proteins described herein and their related variantsare useful in a number of therapeutic and diagnostic applications. Theseinclude the inhibition of the biological activity of PD-L1 by competingfor or blocking the binding to a PD-L1 as well as the delivery ofcytotoxic or imaging moieties to cells, preferably cells expressingPD-L1. The small size and stable structure of these molecules can beparticularly valuable with respect to manufacturing of the drug, rapidclearance from the body for certain applications where rapid clearanceis desired or formulation into novel delivery systems that are suitableor improved using a molecule with such characteristics.

In one aspect, the present disclosure provides methods of treating asubject. The method can, for example, have a generally salubrious effecton the subject, e.g., it can increase the subject's expected longevity.Alternatively, the method can, for example, treat, prevent, cure,relieve, or ameliorate a disease, disorder, condition, or illness (“acondition”). Among the conditions to be treated are conditionscharacterized by inappropriate expression or activity of PD-L1. In somesuch conditions, the expression or activity level is too high, and thetreatment comprises administering a PD-L1 antagonist as describedherein. The disorders or conditions are cancer-related. In particular,those cancers include, but are not limited to, lung, ovarian and coloncarcinoma and various myelomas.

Specific medical conditions and diseases that are treatable orpreventable with the antigen binding proteins of this disclosure includevarious cancers.

On the basis of their efficacy as inhibitors of PD-L1 biologicalactivity, the polypeptides of this disclosure are effective against anumber of cancer conditions as well as complications arising fromcancer, such as pleural effusion and ascites. Preferably, thePD-L1-binding polypeptides of the disclosure can be used for thetreatment of prevention of hyperproliferative diseases or cancer and themetastatic spread of cancers. Preferred indications for the disclosedanti-PD-L1 antibodies include colorectal cancers, head and neck cancers,small cell lung cancer, non-small cell lung cancer (NSCLC) andpancreatic cancer. Non-limiting examples of cancers include bladder,blood, bone, brain, breast, cartilage, colon kidney, liver, lung, lymphnode, nervous tissue, ovary, pancreatic, prostate, skeletal muscle,skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea,urogenital tract, ureter, urethra, uterus, or vaginal cancer. In certainembodiments, the cancer to be treated is selected from the groupconsisting of ovarian cancer, colon cancer, breast cancer or hepaticcarcinoma, myeloma, neuroblastic-derived CNS tumor, monocytic leukemia,B-cell derived leukemia, T-cell derived leukemia, B-cell derivedlymphoma, T-cell derived lymphoma, a mast cell derived tumor, and anycombination thereof.

In certain embodiments, the broad spectrum of mammalian cancers to betreated is selected from the group consisting of ovarian, colon, breast,lung cancers, myelomas, neuroblastic-derived CNS tumors, monocyticleukemias, B-cell derived leukemias, T-cell derived leukemias, B-cellderived lymphomas, T-cell derived lymphomas, mast cell derived tumors,and combinations thereof. Preferably, the autoimmune disease orinflammatory disease is selected from the group consisting of intestinalmucosal inflammation, wasting disease associated with colitis, multiplesclerosis, systemic lupus erythematosus, viral infections, rheumatoidarthritis, osteoarthritis, psoriasis, Crohn's disease, and inflammatorybowel disease.

In addition, various inflammatory disorders can be treated with thedisclosed anti-PD-L1 binding polypeptides disclosed herein. Suchinflammatory disorders include, for example, intestinal mucosainflammation wasting diseases associated with colitis, multiplesclerosis, systemic lupus erythematosus, viral infections, rheumatoidarthritis, osteoarthritis, psoriasis, and Crohn's disease.

Use of antigen binding proteins in ex vivo procedures also iscontemplated. For example, a patient's blood or other bodily fluid maybe contacted with an antigen binding protein that binds PD-L1 ex vivo.The antigen binding protein may be bound to a suitable insoluble matrixor solid support material.

A PD-L1 binding polypeptide can be administered alone or in combinationwith one or more additional therapies such as chemotherapy radiotherapy,immunotherapy, surgical intervention, or any combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described above.

In another embodiment, the method comprises administering one or more ofthe PD-L1 antagonists described herein and one or more other treatments(e.g., a therapeutic or palliative treatment). Where a method comprisesadministering more than one treatment to a subject, it is to beunderstood that the order, timing, number, concentration, and volume ofthe administrations is limited only by the medical requirements andlimitations of the treatment, i.e., two treatments can be administeredto the subject, e.g., simultaneously, consecutively, alternately, oraccording to any other regimen.

Thus, in another aspect, the present disclosure provides a method oftreating a subject with a PD-L1 inhibiting antigen binding protein andone or more other treatments. In one embodiment, such a combinationtherapy achieves synergy or an additive effect by, for example,attacking multiple sites or molecular targets in a tumor. Types ofcombination therapies that can be used in connection with the presentinvention include inhibiting or activating (as appropriate) multiplenodes in a single disease-related pathway, multiple pathways in a targetcell, and multiple cell types within a target tissue.

In another embodiment, a combination therapy method comprisesadministering to the subject two, three, four, five, six, or more of thePD-L1 agonists or antagonists described herein. In another embodiment,the method comprises administering to the subject two or more treatmentsthat together inhibit or activate (directly or indirectly)PD-L1-mediated signal transduction. Examples of such methods includeusing combinations of two or more PD-L1 inhibiting antigen bindingproteins, of a PD-L1 inhibiting antigen binding protein and one or moreother therapeutic moiety having anti-cancer properties (for example,cytotoxic agents, and/or immunomodulators), or of a PD-L1 inhibitingantigen binding protein and one or more other treatments (e.g., surgery,or radiation). Furthermore, one or more anti-PD-L1 antibodies orantibody derivatives can be used in combination with one or moremolecules or other treatments, wherein the other molecule(s) and/ortreatment(s) do not directly bind to or affect PD-L1, but whichcombination is effective for treating or preventing the condition beingtreated. In one embodiment, one or more of the molecule(s) and/ortreatment(s) treats or prevents a condition that is caused by one ormore of the other molecule(s) or treatment(s) in the course of therapy,e.g., nausea, fatigue, alopecia, cachexia, insomnia, etc. In every casewhere a combination of molecules and/or other treatments is used, theindividual molecule(s) and/or treatment(s) can be administered in anyorder, over any length of time, which is effective, e.g.,simultaneously, consecutively, or alternately. In one embodiment, themethod of treatment comprises completing a first course of treatmentwith one molecule or other treatment before beginning a second course oftreatment. The length of time between the end of the first course oftreatment and beginning of the second course of treatment can be anylength of time that allows the total course of therapy to be effective,e.g., seconds, minutes, hours, days, weeks, months, or even years.

In certain embodiments, the subject anti-PD-L1 antibodies agents of theinvention can be used alone. Alternatively, the subject agents may beused in combination with other conventional anti-cancer therapeuticapproaches directed to treatment or prevention of proliferativedisorders (e.g., tumor). For example, such methods can be used inprophylactic cancer prevention, prevention of cancer recurrence andmetastases after surgery, and as an adjuvant of other conventionalcancer therapy. The present disclosure recognizes that the effectivenessof conventional cancer therapies (e.g., chemotherapy, radiation therapy,phototherapy, immunotherapy, and surgery) can be enhanced through theuse of a subject polypeptide therapeutic agent.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

A wide array of conventional compounds have been shown to haveanti-neoplastic activities. These compounds have been used aspharmaceutical agents in chemotherapy to shrink solid tumors, preventmetastases and further growth, or decrease the number of malignant cellsin leukemic or bone marrow malignancies. Although chemotherapy has beeneffective in treating various types of malignancies, manyanti-neoplastic compounds induce undesirable side effects. It has beenshown that when two or more different treatments are combined, thetreatments may work synergistically and allow reduction of dosage ofeach of the treatments, thereby reducing the detrimental side effectsexerted by each compound at higher dosages. In other instances,malignancies that are refractory to a treatment may respond to acombination therapy of two or more different treatments.

When a polypeptide therapeutic agent of the present invention isadministered in combination with another conventional anti-neoplasticagent, either concomitantly or sequentially, such therapeutic agent maybe found to enhance the therapeutic effect of the anti-neoplastic agentor overcome cellular resistance to such anti-neoplastic agent. Thisallows decrease of dosage of an anti-neoplastic agent, thereby reducingthe undesirable side effects, or restores the effectiveness of ananti-neoplastic agent in resistant cells.

Pharmaceutical compounds that may be used for combinatory anti-tumortherapy include, merely to illustrate: aminoglutethimide, amsacrine,anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin,busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine.

Certain chemotherapeutic anti-tumor compounds may be categorized bytheir mechanism of action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (TNP-470, genistein) and growth factorinhibitors (e.g., VEGF inhibitors, fibroblast growth factor (FGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),corticosteroids (cortisone, dexamethasone, hydrocortisone,methylpednisolone, prednisone, and prenisolone); growth factor signaltransduction kinase inhibitors; mitochondrial dysfunction inducers andcaspase activators; and chromatin disruptors.

Depending on the nature of the combinatory therapy, administration ofthe anti-PD-L1 antibody of the invention (or fragment thereof) may becontinued while the other therapy is being administered and/orthereafter. Administration of the polypeptide therapeutic agents may bemade in a single dose, or in multiple doses. In some instances,administration of the polypeptide therapeutic agents is commenced atleast several days prior to the conventional therapy, while in otherinstances, administration is begun either immediately before or at thetime of the administration of the conventional therapy.

In one example of a diagnostic application, a biological sample, such asserum or a tissue biopsy, from a patient suspected of having a conditioncharacterized by inappropriate angiogenesis is contacted with adetectably labeled polypeptide of the disclosure to detect levels ofPD-L1. The levels of PD-L1 detected are then compared to levels of PD-L1detected in a normal sample also contacted with the labeled polypeptide.An increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%in the levels of the PD-L1 may be considered a diagnostic indicator.

In certain embodiments, the PD-L1 binding polypeptides are furtherattached to a label that is able to be detected (e.g., the label can bea radioisotope, fluorescent compound, enzyme or enzyme co-factor). Theactive moiety may be a radioactive agent, such as: radioactive heavymetals such as iron chelates, radioactive chelates of gadolinium ormanganese, positron emitters of oxygen, nitrogen, iron, carbon, orgallium ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ¹²³I, ¹²⁵I, ¹³¹I, ¹³²I or⁹⁹Tc. A binding agent affixed to such a moiety may be used as an imagingagent and is administered in an amount effective for diagnostic use in amammal such as a human and the localization and accumulation of theimaging agent is then detected. The localization and accumulation of theimaging agent may be detected by radioscintigraphy, nuclear magneticresonance imaging, computed tomography or positron emission tomography.Immunoscintigraphy using PD-L1 binding polypeptides directed at PD-L1may be used to detect and/or diagnose cancers and vasculature. Forexample, any of the binding polypeptide against a PD-L1 marker labeledwith ⁹⁹Technetium, ¹¹¹Indium, or ¹²⁵Iodine may be effectively used forsuch imaging. As will be evident to the skilled artisan, the amount ofradioisotope to be administered is dependent upon the radioisotope.Those having ordinary skill in the art can readily formulate the amountof the imaging agent to be administered based upon the specific activityand energy of a given radionuclide used as the active moiety. Typically0.1-100 millicuries per dose of imaging agent, preferably 1-10millicuries, most often 2-5 millicuries are administered. Thus,compositions according to the present invention useful as imaging agentscomprising a targeting moiety conjugated to a radioactive moietycomprise 0.1-100 millicuries, in some embodiments preferably 1-10millicuries, in some embodiments preferably 2-5 millicuries, in someembodiments more preferably 1-5 millicuries.

The anti-PD-L1 antibody of the invention (or fragment thereof) can alsobe used to deliver additional therapeutic agents (including but notlimited to drug compounds, chemotherapeutic compounds, andradiotherapeutic compounds) to a cell or tissue expressing PD-L1. In oneexample, the anti-PD-L1 antibody of the invention (or fragment thereof)is fused to a chemotherapeutic agent for targeted delivery of thechemotherapeutic agent to a tumor cell or tissue expressing PD-L1.

The anti-PD-L1 antibody of the invention (or fragment thereof) areuseful in a variety of applications, including research, diagnostic andtherapeutic applications. For instance, they can be used to isolateand/or purify receptor or portions thereof, and to study receptorstructure (e.g., conformation) and function.

In certain aspects, the various binding polypeptides can be used todetect or measure the expression of PD-L1, for example, on endothelialcells (e.g., venous endothelial cells), or on cells transfected with aPD-L1 gene. Thus, they also have utility in applications such as cellsorting and imaging (e.g., flow cytometry, and fluorescence activatedcell sorting), for diagnostic or research purposes.

In certain embodiments, the binding polypeptides of fragments thereofcan be labeled or unlabeled for diagnostic purposes. Typically,diagnostic assays entail detecting the formation of a complex resultingfrom the binding of a binding polypeptide to PD-L1. The bindingpolypeptides or fragments can be directly labeled, similar toantibodies. A variety of labels can be employed, including, but notlimited to, radionuclides, fluorescers, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (U.S.Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and 4,098,876). Whenunlabeled, the binding polypeptides can be used in assays, such asagglutination assays. Unlabeled binding polypeptides can also be used incombination with another (one or more) suitable reagent which can beused to detect the binding polypeptide, such as a labeled antibodyreactive with the binding polypeptide or other suitable reagent (e.g.,labeled protein A).

In one embodiment, the binding polypeptides of the present invention canbe utilized in enzyme immunoassays, wherein the subject polypeptides areconjugated to an enzyme. When a biological sample comprising a PD-L1protein is combined with the subject binding polypeptides, bindingoccurs between the binding polypeptides and the PD-L1 protein. In oneembodiment, a sample containing cells expressing a PD-L1 protein (e.g.,endothelial cells) is combined with the subject antibodies, and bindingoccurs between the binding polypeptides and cells bearing a PD-L1protein recognized by the binding polypeptide. These bound cells can beseparated from unbound reagents and the presence of the bindingpolypeptide-enzyme conjugate specifically bound to the cells can bedetermined, for example, by contacting the sample with a substrate ofthe enzyme which produces a color or other detectable change when actedon by the enzyme. In another embodiment, the subject bindingpolypeptides can be unlabeled, and a second, labeled polypeptide (e.g.,an antibody) can be added which recognizes the subject bindingpolypeptide.

In certain aspects, kits for use in detecting the presence of a PD-L1protein in a biological sample can also be prepared. Such kits willinclude a PD-L1 binding polypeptide which binds to a PD-L1 protein orportion of said receptor, as well as one or more ancillary reagentssuitable for detecting the presence of a complex between the bindingpolypeptide and the receptor protein or portions thereof. Thepolypeptide compositions of the present invention can be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes. The binding polypeptides and/orantibodies, which can be labeled or unlabeled, can be included in thekits with adjunct ingredients (e.g., buffers, such as Tris, phosphateand carbonate, stabilizers, excipients, biocides and/or inert proteins,e.g., bovine serum albumin). For example, the binding polypeptidesand/or antibodies can be provided as a lyophilized mixture with theadjunct ingredients, or the adjunct ingredients can be separatelyprovided for combination by the user. Generally these adjunct materialswill be present in less than about 5% weight based on the amount ofactive binding polypeptide or antibody, and usually will be present in atotal amount of at least about 0.001% weight based on polypeptide orantibody concentration. Where a second antibody capable of binding tothe binding polypeptide is employed, such antibody can be provided inthe kit, for instance in a separate vial or container. The secondantibody, if present, is typically labeled, and can be formulated in ananalogous manner with the antibody formulations described above.

Similarly, the present disclosure also provides a method of detectingand/or quantitating expression of PD-L1, wherein a compositioncomprising a cell or fraction thereof (e.g., membrane fraction) iscontacted with a binding polypeptide which binds to a PD-L1 or portionof the receptor under conditions appropriate for binding thereto, andthe binding is monitored. Detection of the binding polypeptide,indicative of the formation of a complex between binding polypeptide andPD-L1 or a portion thereof, indicates the presence of the receptor.Binding of a polypeptide to the cell can be determined by standardmethods, such as those described in the working examples. The method canbe used to detect expression of PD-L1 on cells from an individual.Optionally, a quantitative expression of PD-L1 on the surface ofendothelial cells can be evaluated, for instance, by flow cytometry, andthe staining intensity can be correlated with disease susceptibility,progression or risk.

The present disclosure also provides a method of detecting thesusceptibility of a mammal to certain diseases. To illustrate, themethod can be used to detect the susceptibility of a mammal to diseaseswhich progress based on the amount of PD-L1 present on cells and/or thenumber of PD-L1-positive cells in a mammal.

Polypeptide sequences are indicated using standard one- or three-letterabbreviations. Unless otherwise indicated, each polypeptide sequence hasamino termini at the left and a carboxy termini at the right; eachsingle-stranded nucleic acid sequence, and the top strand of eachdouble-stranded nucleic acid sequence, has a 5′ termini at the left anda 3′ termini at the right. A particular polypeptide sequence also can bedescribed by explaining how it differs from a reference sequence.

Antigen binding proteins directed against PD-L1 can be used, forexample, in assays to detect the presence of PD-L1 polypeptides, eitherin vitro or in vivo. The antigen binding proteins also may be employedin purifying PD-L1 proteins by immunoaffinity chromatography. Blockingantigen binding proteins can be used in the methods disclosed herein.Such antigen binding proteins that function as PD-L1 antagonists may beemployed in treating any PD-L1-induced condition, including but notlimited to various cancers.

Antigen binding proteins may be employed in an in vitro procedure, oradministered in vivo to inhibit PD-L1-induced biological activity.Disorders caused or exacerbated (directly or indirectly) by theproteolytic activation of PD-L1, examples of which are provided herein,thus may be treated. In one embodiment, the present invention provides atherapeutic method comprising in vivo administration of a PD-L1 blockingantigen binding protein to a mammal in need thereof in an amounteffective for reducing an PD-L1-induced biological activity.

Pharmaceutical Formulations of Disclosed Antibodies with Tumor Vaccines

A combined therapeutic product or formulation of a disclosed anti-PD-L1antibody with a therapeutic vaccine provides synergistic oncologictherapeutic benefit. For example, the present disclosure provides acombination of a disclosed anti-PD-L1 antibody with “Neuvax” which is aE75-derived 9 mer synthetic peptide isolated from HER2/neu combined withGM-CSF as an adjuvant as described in U.S. Pat. No. 8,222,214, thedisclosure of which is incorporated by reference herein. In addition,the present disclosure provides a combination of a disclosed anti-PD-L1antibody with ALVAC-CEA vaccine, which is a canary pox virus combinedwith carcinoembryonic antigen.

Production of Anti-PD-L1 Antigen Binding Proteins

Antigen binding proteins may be prepared by any of a number ofconventional techniques. The present disclosure provides, in oneembodiment, monoclonal antibodies that bind to PD-L1. Monoclonalantibodies may, for example, be purified from cells that naturallyexpress them (e.g., an antibody can be purified from a hybridoma thatproduces it), or produced in recombinant expression systems, using anytechnique known in the art. See, for example, Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.), Plenum Press, New York (1980); and Antibodies: A LaboratoryManual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988).

Monoclonal antibodies may be produced using any technique known in theart, e.g., by immortalizing spleen cells harvested from the transgenicanimal after completion of the immunization schedule. The spleen cellscan be immortalized using any technique known in the art, e.g., byfusing them with myeloma cells to produce hybridomas. Myeloma cells foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Examples of suitable cell lines for use in mouse fusionsinclude Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; examples of celllines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and48210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6.

In one example, the polypeptides are produced by recombinant DNA methodsby inserting a nucleic acid sequence (e.g., a cDNA) encoding thepolypeptide into a recombinant expression vector and expressing the DNAsequence under conditions promoting expression.

For recombinant production of an anti-PD-L1 antibody, nucleic acidencoding an antibody is isolated and inserted into one or more vectorsfor further cloning and/or expression in a host cell. Such nucleic acidmay be readily isolated and sequenced using conventional procedures(e.g., by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of theantibody).

Nucleic acids encoding any of anti-PD-L1 antibodies (or fragments)disclosed herein may be synthesized chemically. Codon usage may beselected so as to improve expression in a cell. Such codon usage willdepend on the cell type selected. Specialized codon usage patterns havebeen developed for E. coli and other bacteria, as well as mammaliancells, plant cells, yeast cells and insect cells. See for example:Mayfield et al., Proc. Natl. Acad. Sci. USA. 2003 100(2):438-42;Sinclair et al. Protein Expr. Purif. 2002 (1):96-105; Connell N D. Curr.Opin. Biotechnol. 2001 12(5):446-9; Makrides et al. Microbiol. Rev. 199660(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants, is additionally incorporated.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).

The expression construct is introduced into the host cell using a methodappropriate to the host cell. A variety of methods for introducingnucleic acids into host cells are known in the art, including, but notlimited to, electroporation; transfection employing calcium chloride,rubidium chloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent). Suitable host cells include prokaryotes,yeast, mammalian cells, or bacterial cells, as described in more detailbelow.

Any expression system known in the art can be used to make therecombinant polypeptides (e.g., recombinant antibody) of the invention.In general, host cells are transformed with a recombinant expressionvector that comprises DNA encoding a desired polypeptide. Among the hostcells that may be employed are prokaryotes, yeast or higher eukaryoticcells. Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Higher eukaryotic cells include insect cellsand established cell lines of mammalian origin. Suitable host cells forcloning or expression of antibody-encoding vectors include prokaryoticor eukaryotic cells described herein. For example, antibodies may beproduced in bacteria, in particular when glycosylation and Fc effectorfunction are not needed. For expression of antibody fragments andpolypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,5,789,199, and 5,840,523. (See also Charlton, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003),pp. 245-254, describing expression of antibody fragments in E. coli.)After expression, the antibody may be isolated from the bacterial cellpaste in a soluble fraction and can be further purified. Highereukaryotic cells include insect cells and established cell lines ofmammalian origin. Examples of suitable mammalian host cell lines includethe COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al.,1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10)cell lines, and the CV1/EBNA cell line derived from the African greenmonkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan etal., 1991, EMBO J. 10: 2821. Appropriate cloning and expression vectorsfor use with bacterial, fungal, yeast, and mammalian cellular hosts aredescribed by Pouwels et al. (Cloning Vectors: A Laboratory Manual,Elsevier, N.Y., 1985).

In certain embodiments, vertebrate cells may be used as hosts to expressan anti-PD-L1 antibody or fragment thereof. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR.sup.-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003). Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK(ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from theAfrican green monkey kidney cell line CV1 (ATCC CCL 70) as described byMcMahan et al., 1991, EMBO J. 10: 2821.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described by Pouwels etal. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having all or a portion (e.g., the extracellular domain) of PD-L1bound thereto. Polypeptides contemplated for use herein includesubstantially homogeneous recombinant mammalian anti-PD-L1 antibodypolypeptides substantially free of contaminating endogenous materials.

Thus, antibodies may be produced using recombinant methods andcompositions, e.g., as described in U.S. Pat. No. 4,816,567. In oneembodiment, isolated nucleic acid encoding an anti-PD-L1 antibodydescribed herein is provided. Such nucleic acid may encode an amino acidsequence comprising the VL and/or an amino acid sequence comprising theVH of the antibody (e.g., the light and/or heavy chains of theantibody). In a further embodiment, one or more vectors (e.g.,expression vectors) comprising such nucleic acid are provided. In afurther embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In oneembodiment, a method of making an anti-PD-L1 antibody is provided,wherein the method comprises culturing a host cell comprising a nucleicacid encoding the antibody, as provided above, under conditions suitablefor expression of the antibody, and optionally recovering the antibodyfrom the host cell (or host cell culture medium).

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system.

PD-L1-binding polypeptides can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications tothe protein can also be produced by chemical synthesis.

The polypeptides of the present disclosure can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, morepreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the polypeptideis sufficiently pure for use as a pharmaceutical product. Antigenbinding proteins (e.g., antibodies, antibody fragments, antibodyderivatives, antibody muteins, and antibody variants) are polypeptidesthat bind to PD-L1, (preferably, human PD-L1). Antigen binding proteinsinclude antigen binding proteins that inhibit a biological activity ofPD-L1.

Antigen binding proteins may be prepared, and screened for desiredproperties, by any of a number of known techniques. Certain of thetechniques involve isolating a nucleic acid encoding a polypeptide chain(or portion thereof) of an antigen binding protein of interest (e.g., ananti-PD-L1 antibody), and manipulating the nucleic acid throughrecombinant DNA technology. The nucleic acid may be fused to anothernucleic acid of interest, or altered (e.g., by mutagenesis or otherconventional techniques) to add, delete, or substitute one or more aminoacid residues, for example.

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol. Biol. 178:379-87.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype (Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover,if an IgG4 is desired, it may also be desired to introduce a pointmutation (CPSCP→CPPCP) in the hinge region (Bloom et al., 1997, ProteinScience 6:407) to alleviate a tendency to form intra-H chain disulfidebonds that can lead to heterogeneity in the IgG4 antibodies.

Other embodiments are described in the following non-limiting Examples.

Example 1: Comparative Affinity for H6B1L Antibody and VariantAntibodies

This example provides Biacore data showing that the three fully humanantibodies, wild type (H6B1L), H6B1L-EV and H6B1L-EM have similaraffinity to each other to human PD-L1, as shown in Table 1 below:

TABLE 1 Comparative BIACORE data for parent antibody H6B1L and variantsH6B1L-EV and -EM Rmax Name ka (1/Ms) kd (1/s) (RU) KA (1/M) KD (M) Chi2H6B1L 1.51E+06 2.80E−03 60.1 5.40E+08 1.85E−09 0.38 H6B1L- 1.57E+062.87E−03 57.3 5.48E+08 1.82E−09 0.496 EV H6B1L- 1.38E6 2.14E−03 2486.44E+08 1.55E−09 7.93 EM

Accordingly, surprisingly, changing one or two amino acids in the lightchain improved the ability to manufacture the antibody in sufficientquantities but did not impact the binding ability to bind to its target,human PD-L1.

Example 2: Improved Manufacturing of Antibody H6B1L-EM

This example illustrates a mass spectral analysis of two light chainsfrom the parent wild type antibody H6B1L (SEQ ID NO. 4) and H6B1L-EMlight chain (SEQ ID NO. 2). The antibodies were each produced in CHOcells. A comparison of the peak achieved is shown in FIG. 1A. Inaddition, N-terminal Edman sequencing confirmed that there is a lightchain fragment of SYELMXXX and LMXXX present in the mass spec peaks forwild type H6B1L light chain. As described in FIG. 1B, no light chain(LC) fragment was detected for the H6B1LEM light chain in comparison tothe parent light chain.

SEQUENCE TABLE Light chain variable domain region Changes relative toH6B1L parent chain are un- derlined. Bold residues inthe H6B1L chain show muta- tion positions in variantHeavy chain variable domain chains EM and EV. Italicizedregion. Italicized residues residues indicate CDRindicate CDR sequences. sequences. H6B1L-EM QMQLVQSGAEVKKPGSSVKVSCKASSYVLTQPPSVSVAPGKTATIACGGENIGR GGTFSSYAYSWVRQAPGQGLEWMGGKTVHWYQQKPGQAPVLVIYYDSDRPSGI IIPSFGTANYAQKFQGRVTITADESTSTPERFSGSNSGNTATLTISRVEAGDEADY AYMELSSLRSEDTAVYYCARGPIVATYCLVWDSSSDHRIFGGGTKLTVL ITPLDYWGQGTLVTVSS (SEQ ID NO. 2) (SEQ ID NO. 1)H6B1L-EV QMQLVQSGAEVKKPGSSVKVSCKAS SYVLMQPPSVSVAPGKTATIACGGENIGGGTFSSYAYSWVRQAPGQGLEWMGG RKTVHWYQQKPGQAPVLVIYYDSDRPSGIIPSFGTANYAQKFQGRVTITADESTST IPERFSGSNSGNTATLTISRVEAGDEADYAYMELSSLRSEDTAVYYCARGPIVAT YCLVWDSSSDHRIFGGGTKLTVL ITPLDYWGQGTLVTVSS(SEQ ID NO. 3) (SEQ ID NO. 1) H6B1L QMQLVQSGAEVKKPGSSVKVSCKASSYELMQPPSVSVAPGKTATIACGGENIG GGTFSSYAYSWVRQAPGQGLEWMGGRKTVHWYQQKPGQAPVLVIYYDSDRPSG IIPSFGTANYAQKFQGRVTITADESTSTIPERFSGSNSGNTATLTISRVEAGDEADY AYMELSSLRSEDTAVYYCARGPIVATYCLVWDSSSDHRIFGGGTKLTVL ITPLDYWGQGTLVTVSS (SEQ ID NO. 4) (SEQ ID NO. 1)Heavy Chain SYAYS (SEQ ID NO. 5) Variable Domain CDR1 Heavy ChainGIIPSFGTANYAQKFQG (SEQ ID NO. 6) Variable Domain CDR2 Heavy ChainGPIVATITPLDY (SEQ ID NO. 7) Variable Domain CDR3 Light ChainGGENIGRKTVH (SEQ ID NO. 8) Variable Domain CDR1 Light ChainYDSDRPS (SEQ ID NO. 9) Variable Domain CDR2 Light ChainLVWDSSSDHRI (SEQ ID NO. 10) Variable Domain CDR3

INCORPORATION BY REFERENCE

The contents of all references, patents, and patent applications citedthroughout this application are hereby expressly incorporated byreference.

I claim:
 1. A fully human antibody of an IgG class that binds to a PD-L1 epitope, wherein the antibody comprises a heavy chain variable domain comprising a CDR1 domain, a CDR2 domain, and a CDR3 domain as set forth in the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and comprises a light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO. 2 or SEQ ID NO.
 3. 2. The fully human antibody of claim 1, wherein the antibody has a heavy chain/light chain variable domain amino acid sequence combination of either SEQ ID NO. 1/SEQ ID NO. 2 or SEQ ID NO. 1/SEQ ID NO.
 3. 3. The fully human antibody of claim 1, which is an IgG1 or an IgG4.
 4. An anti-PD-L1 Fab fully human antibody fragment having a variable domain from a heavy chain and a variable domain from a light chain, wherein the heavy chain variable domain comprises a CDR1 domain, a CDR2 domain, and a CDR3 domain as set forth in the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and wherein the light chain variable domain comprises an amino acid sequence as set forth in SEQ ID NO. 2 or SEQ ID NO.
 3. 5. The fully human antibody Fab fragment of claim 4, wherein the antibody has a heavy chain/light chain variable domain amino acid sequence combination of either SEQ ID NO. 1/SEQ ID NO. 2 or SEQ ID NO. 1/SEQ ID NO.
 3. 6. An anti-PD-L1 single chain human antibody having a heavy chain variable domain and a light chain variable domain connected via a peptide linker, wherein the heavy chain variable domain comprises a CDR1 domain, a CDR2 domain, and a CDR3 domain as set forth in the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and wherein the light chain variable domain comprises an amino acid sequence as set forth in either SEQ ID NO. 2 or SEQ ID NO.
 3. 7. The fully human single chain antibody of claim 6, wherein the single chain fully human antibody has a heavy chain/light chain variable domain amino acid sequence combination of either SEQ ID NO. 1/SEQ ID NO. 2 or SEQ ID NO. 1/SEQ ID NO.
 3. 8. A method for treating a human subject having cancer or an autoimmune or inflammatory disease, said method comprising administering an effective amount of the fully human antibody of claim 1 to the human subject.
 9. A method for treating a human subject having cancer or an autoimmune or inflammatory disease, said method comprising administering an effective amount of the fully human antibody Fab fragment of claim 4 to the human subject.
 10. A method for treating a human subject having cancer or an autoimmune or inflammatory disease, said method comprising administering an effective amount of the fully human single chain antibody of claim 6 to the human subject.
 11. The method of claim 8, wherein the cancer is selected from the group consisting of ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, a neuroblastic-derived CNS tumor, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, B-cell derived lymphoma, T-cell derived lymphoma, and a mast cell derived tumor.
 12. The method of claim 8, wherein the autoimmune or inflammatory disease is selected from the group consisting of intestinal mucosal inflammation, wasting disease associated with colitis, multiple sclerosis, systemic lupus erythematosus, a viral infection, rheumatoid arthritis, osteoarthritis, psoriasis, Crohn's disease, and inflammatory bowel disease.
 13. The antibody of claim 1, which is produced in a mammalian host cell.
 14. The antibody of claim 13, wherein the mammalian host cell is a Chinese hamster ovary (CHO) cell.
 15. A pharmaceutical composition comprising the fully human antibody of claim 1, and a pharmaceutically acceptable excipient.
 16. A pharmaceutical composition comprising the fully human antibody Fab fragment of claim 4, and a pharmaceutically acceptable excipient.
 17. A pharmaceutical composition comprising the fully human single chain antibody of claim 6, and a pharmaceutically acceptable excipient. 